show Abstracthide AbstractHydrogenases are a critical component of H2-dependent energy-conservation pathways in Methanosarcina barkeri. To allow phenotypic analysis of the hydrogenases, we constructed mutants lacking the frhADGB, freAEGB, vhtGACD, vhxGAC and echABCDEF operons, individually and in all possible combinations. In addition to measuring the effect of each deletion on growth, methane production, and hydrogenase activity, the effect on gene expression was measured by RNA sequencing to detect potential transcriptional regulation by the hydrogenases. Overall design: The parental strain of M. barkeri and strains in which the hydrogenases were removed in various combinations were grown to mid-exponential phase on media containing methanol as the growth substrate. RNA was extracted, sequenced, and mapped to the M. barkeri genome. Pairwise comparisons of transcript levels were made for each strain against all other strains.